Genetic evaluation of physiological functions of thiolase isoenzymes in the n-alkalane-assimilating yeast Candida tropicalis.
The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1a delta/t1bdelta null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3a delta/t3b delta null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C10 to C13). All thiolase activities in this yeast disappeared with the ct-t1a delta/t1bdelta and ct-t3a delta/t3bdelta null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bdelta null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal beta-oxidation. In addition to its essential contribution to the beta-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the beta-oxidation of a C4 substrate butyrate.
Kanayama
N
N
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
Ueda
M
M
Atomi
H
H
Tanaka
A
A
D000101:Acetyl-CoA C-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000102:Acetyl-CoA C-Acyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000473:Alkanes / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004271:DNA, Fungal; D015870:Gene Expression; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid
D000473:Alkanes; D004271:DNA, Fungal; D007527:Isoenzymes; D000102:Acetyl-CoA C-Acyltransferase; D000101:Acetyl-CoA C-Acetyltransferase; D008798:Mevalonic Acid